Notch signaling influences development and maintenance of the nervous system through directing the specification of neural progenitors into neurons and glia, as well as regulating neurite morphogenesis and synaptic interactions. Notch was first identified as a neurogenic gene, yet activation of Notch receptors by Delta/Serrate/Lag2 (DSL) ligands regulates the differentiation of a wide variety of neuronal and non-neuronal cell types. Moreover, Notch signaling has been linked to certain cancers, inherited human syndromes and neurodegenerative diseases. Although a role for Notch in these different processes is certain, the molecular mechanisms of ligand-induced Notch signaling are not well defined. To biochemically and molecularly characterize ligand-Notch interactions and downstream signaling events we have developed ligand-binding and cell co-culture assays in which components of the Notch signaling system can be readily manipulated and studied. Importantly, these co-culture systems measure Notch signaling induced by ligand, which is more reflective of physiological Notch signaling than the widely used constitutively active forms of Notch. Our studies have identified a noncanonical Notch signaling pathway that differs from the "core" Notch signaling pathway both in the Notch isoform and downstream effector, CSL (CBF-1, Su(H), Lag-1), activated by ligand, in addition, we have identified sequences within both the DSL ligand and Notch receptor proteins that confer ligand-receptor specificity and have demonstrated that the glycosyltransferase fringe differentially modulates ligand-induced Notch signaling as first described for Drosophila. Finally, we have discovered that the DSL family member, Delta3, does not activate Notch signaling but rather functions to inhibit ligand-induced Notch signaling. The experiments proposed in this application are logical extensions of our previous findings. Three aims are proposed to (1) define the requirements and downstream targets of CSL-independent Notch signaling; (2) characterize the effects of different ligand-receptor combinations on Notch signaling; and (3) characterize the inhibitory effects of Delta3 on ligand-induced Notch signaling. Information obtained using these approaches will extend our current understanding of the molecular mechanisms of Notch signal transduction and provide clues as to how this signaling system regulates a diverse array of cellular processes both in the embryo and in the adult.